Isolation and Characterization of Antibiotics from Actinomycetes Collected in Nepal
Date
2024-07
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Publisher
Institute of Science & Technology
Abstract
Actinomycetes are a distinct class of Gram-positive bacteria with a filamentous mycelium-like that of fungi. They produce many potential antitumor, anticancer, and antimicrobials; a key source of biologically active compounds to combat infectious diseases and others. Moreover, multi-drug resistant (MDR) pathogens cause serious problems in both humans and animals. This issue demands a need for stronger antimicrobial agents. This research has examined the secondary metabolites produced by soil-based actinomycetes collected in various parts of Nepal.
Actinomycetes were isolated from soils by using the International Streptomyces Project 4 (ISP4) medium. Most of the aerial mycelia of bacterial isolates had a greyish-white color whereas some of them appeared bluish-white. The prominent colonies of isolated bacteria were cultured in a Tryptic Soy Broth (TSB) medium. The Gram-positive nature of the isolated bacteria was confirmed by Gram staining.
Ethyl acetate extracts of the isolates were prepared after shake flask fermentation in the TSB medium. The antimicrobial activity of the isolates was carried out against Staphylococcus aureus American Type Culture Collection (ATCC) 43300, Shigella sonnei ATCC 25931, Salmonella typhi ATCC 14028, Klebsiella pneumoniae ATCC 700603 and Escherichia coli ATCC 25922 bacteria, and observed through primary and secondary microbiological screening. Primary screening of the isolates was performed by the perpendicular-streaking method. Most of the isolates showed strong antimicrobial activity against the tested pathogens. A total of 11 bacterial isolates exhibited significant zones of inhibition against both Gram-positive and Gram-negative pathogens. Isolates BT1, BT2, BT3, AB1, PT7, and PC1 showed greater antimicrobial efficacy against tested pathogens based on their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Isolates BT5, BT13, BT33, BT36, and BT39 exhibited remarkable antimicrobial efficacy against tested pathogens. Among the isolates, isolate PC1 showed the highest zone of inhibition (32 mm. against E. coli, 30 mm. against S. typhi, 30 mm. against S. sonnei, and 30 mm. against K. pneumoniae) in all tested pathogens except in S. aureus.
The genomic deoxyribonucleic acid (DNA) of the isolated bacteria was extracted using the standard protocol of molecular biology. The amplified polymerase chain reaction (PCR) product was then subjected to 16S Ribosomal ribonucleic acid (rRNA) sequencing. As such this study identified five Streptomyces species (Streptomyces sp.BT1, Streptomyces sp. BT2, Streptomyces sp. BT3, Streptomyces sp. BT5 and Streptomyces sp. PC1).
The metabolic profiles of the ethyl acetate extracts were investigated using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Fifty-eight compounds were annotated from the raw data files of tandem mass using MestReNova (Mnova) software. Then, the annotated compounds were further validated through SIRIUS software. Out of the annotated compounds from LC-HRMS/MS, four of them were detected for the first time in Streptomyces species, namely Cyclo(Ile-Ser) from isolate BT2, 2-n-hexyl-5-n-propylresorcinol from isolate BT1, 3-((6-methylpyrazin-2-yl)methyl)-1H-indole from isolate PC1, and Cyclo(D-Leu-L-Trp) from isolate BT2. Total 7 antibiotics were annotated which were myxopyronin B, blasticidin H, flavofungin, xenocoumacin 2, okilactomycin A, butyrolactol A and SF2415-B2.
Accordingly, this research identified that Nepal's varied ecology offers ideal circumstances for the existence of several Streptomyces species from the soil habitat which can produce specialized secondary metabolites including antibiotics. This study set up a platform in Nepal, which inspired for isolation of rare actinomycetes, which could produce new therapeutics including antibiotics.
Description
Keywords
Actinomycetes, Multi-drug, Resistant, Secondary Metabolites, Primary Screening, Mass Spectrometry, Streptomyces Species